Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the United States, with a five-year survival rate of less than 5%, and a median survival after diagnosis of 6 months. These statistics reflect the lack of effective early detection and the absence of effective treatments for systemic disease. Greater knowledge of the underlying biology of PDAC initiation and progression is required before more targeted and effective therapies can be developed. The proposed work will explore the potential relationship between the driving mutation of PDAC (mutationally activated KrasG12D) and the miR-17~92 cluster of microRNAs, specifically addressing the requirement for miR-17~92 in Kras' proliferative, pro-survival and tumorigenic phenotypes within the pancreas and the molecular mechanism behind this relationship. To determine whether miR-17~92 miRNAs are required for KrasG12D-induced cellular phenotypes, primary ductal epithelial cells (PDECs) will be isolated and the effect of miR-17~92 deletion on KrasG12D-induced proliferation and survival in the presence of apoptotic stimuli will be determined. Components of the miRNA cluster will be expressed exogenously to assess potential rescue of these phenotypes by specific miRNAs. The effect of deletion of the miR-17~92 cluster on PDAC precursor lesion formation in vivo will be assessed in animals that endogenously express KrasG12D and have deleted the miR-17~92 cluster specifically in the pancreas. Animals will be sacrificed at 4, 8, and 12 months of age and histologic sections of the pancreas examined for the presence, quantity and grade of precursor lesions. Using a mouse model that additionally incorporates heterozygous deletion of the TP53 tumor suppressor gene, the impact of miR-17~92 deletion on PDAC development and progression will be ascertained. These studies are the focus of Specific Aim 1. This study will also identify the mechanism of interaction between the miR-17~92 cluster and activated Kras. Through the use of multiple target prediction algorithms and data from a messenger RNA microarray profile of PDECs expressing KrasG12D, probable targets of the miR-17~92 cluster that are expressed in PDECs will be identified. Expression levels will be evaluated in miR-17~92 wild type and null PDECs by Western blot, and miRNA-mediated suppression will be confirmed by fusing target 3'UTRs to luciferase and measuring luminescence in cells with and without the miRNA cluster. Downstream pathway activity of Kras will be measured in cluster-null PDECs to address whether 17~92 modulates KrasG12D-induced phenotypes through the regulation of specific downstream effector pathways. These studies are the focus of Specific Aim 2. Together, the studies proposed in this application will shed light on the role of the miR-17~92 miRNA cluster in pancreatic cancer initiation and progression, potentially laying the foundation for the development of novel therapeutic approaches.